The nature and function of any given cell depends on the particular set of genes being expressed (e.g., transcribed and translated). Thus, development of pluripotent or totipotent cells into a differentiated, specialized phenotype is determined by the particular genes expressed during development. Gene expression is directly mediated by sequence-specific binding of gene regulatory proteins that can effect either positive or negative regulation. However, the ability of any of these regulatory proteins to directly mediate gene expression appears to depends on the accessibility of their binding site within the cellular DNA. Accessibility of sequences in cellular DNA often depends on the structure of cellular chromatin within which cellular DNA is packaged.
Cellular DNA, including the cellular genome, generally exists in the form of chromatin, a complex comprising nucleic acid and protein. Indeed, most cellular RNAs also exist in the form of nucleoprotein complexes. The nucleoprotein structure of chromatin has been the subject of extensive research, as is known to those of skill in the art. In general, chromosomal DNA is packaged into nucleosomes. A nucleosome comprises a core and a linker. The nucleosome core comprises an octamer of core histones (two each of H2A, H2B, H3 and H4) around which is wrapped approximately 150 base pairs of chromosomal DNA. In addition, a linker DNA segment of approximately 50 base pairs is associated with linker histone H1. Nucleosomes are organized into a higher-order chromatin fiber and chromatin fibers are organized into chromosomes. See, for example, Wolffe “Chromatin: Structure and Function” 3rd Ed., Academic Press, San Diego, 1998.
The structure of chromatin can be altered through the activity of macromolecular assemblies known as chromatin remodeling complexes. See, for example, Cairns (1998) Trends Biochem. Sci. 23:20–25; Workman et al. (1998) Ann. Rev. Biochem. 67:545–579; Kingston et al. (1999) Genes Devel. 13:2339–2352 and Murchardt et al. (1999) J. Mol. Biol. 293:185–197. Recently, chromatin remodeling complexes have been implicated in the disruption or reformation of nucleosomal arrays, resulting in modulation of transcription, DNA replication, and DNA repair. Bochar et al. (2000) PNAS USA 97(3): 1038–43. Many of these chromatin remodeling complexes have different subunit compositions, but all rely on ATPase enzymes for remodeling activity. There are also several examples of a requirement for the activity of chromatin remodeling complexes for gene activation in vivo. The human SWI/SNF chromatin remodeling complex is required for the activity of the glucocorticoid receptor. Fryer et al. (1998) Nature 393:88–91. The mammalian SWI/SNF chromatin remodeling complex is required for activation of the hsp70 gene. de La Serna et al (2000) Mol. Cell. Biol. 20:2839–2851. In addition, mutations in the yeast SWI/SNF gene result in a decrease in expression of one group of genes and an increase in expression of another group of genes, showing that chromatin remodeling can have both positive and negative effects on gene expression. Holsteege et al. (1998) Cell 95:717–728; Sudarsanam et al. (2000) Proc. Natl. Acad. Sci. USA 97:3364–3369.
It has also been shown that mutations in the Drosophila ISWI protein adversely affect expression of the engrailed and Ultrabithorax genes. Deuring et al. (2000) Mol. Cell 5:355–365. The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin (Corona et al. (1999) Mol Cell 3(2):239–245). Isolated ISWI protein has been shown to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions (Corona et al., supra). The ATPase activity of ISWI is stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling by ISWI, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes.
Successful cloning of frogs and several mammalian species by nuclear transplantation has been reported (Gurdon (1962) Dev Biol 4:256–273; Wilmut et al. (1997) Nature 385:810–813; Wakayama et al. (1998) Nature 394:369–374; Kato et al. (1998) Science 282:2095–2098), establishing the remarkable reversibility of the genetic and epigenetic programs that define cell differentiation. Even highly differentiated somatic nuclei can dedifferentiate in egg cytoplasm to acquire the totipotency essential to support normal development to reproductive adulthood. However, despite the ability to generate adult animals from transplanted somatic nuclei, the process is extremely inefficient. See, e.g., Gurdon et al. (1999) Nature 402:743–746. Methods and compositions to increase the efficiency of cloning by nuclear transplantation would clearly advance the field.
The ability of a somatic nucleus to program the development of an entire organism indicates that a somatic nucleus can be dedifferentiated in the egg cytoplasm. Patterns of gene expression in embryos derived from adult nuclei several hours after their transplantation into eggs are indistinguishable from the patterns of normal embryos at the same developmental stage. Chan et al. (1996) Intl. J. Dev. Biol. 40:441–451. This result indicates that nuclear reprogramming is manifested, at least in part, by changes in gene expression that likely result from the change in cytoplasmic environment experienced by the somatic nucleus. This dedifferentiation of a somatic nucleus by egg cytoplasm may be mediated by gain and/or loss of proteins which directly or indirectly affect gene expression. Indeed, somatic nuclei transplanted into Xenopus eggs lose more than 85% of radiolabeled nuclear protein concomitant with substantial uptake of proteins from the egg cytoplasm (Gurdon et al. (1976) J Embryol Exp Morphol 36:541–553; Gurdon et al. (1979) Int Rev Cyto Suppl 9:161–178). Transplanted nuclei might lose preexisting chromatin binding proteins passively through dilution during DNA replication in the comparatively large volume of the egg/embryo cytoplasm. However, emerging evidence suggests that reprogramming of epigenetic states can occur in the nuclei of differentiated cells following heterokaryon formation with stem cells, a process in which dilution does not occur (Tada et al. (1997) EMBO J 16:6510–6520). Thus, it is possible that nuclear reprogramming is mediated by remodeling of nuclear chromatin. In support of this idea, it has been found that both local remodeling of chromatin and alterations in DNA methylation states, events which result in dramatic changes in the functionality of DNA, leading to alterations in gene activity and cell physiology, require the activity of nucleosomal ATPases (Jeddeloh et al. (1999) Nat Genet 22:94–97).
Whether differentiated nuclei undergo specific or global chromatin remodeling activities upon transplantation into eggs, and/or whether chromatin remodeling occurs during generation of stem cell lineages is not well studied. Gurdon et al. (1999) supra. It is possible that some sort of reprogramming or global chromatin remodeling would increase the success rate of cloning by nuclear transplantation Thus, new methods of gene regulation and nuclear reprogramming, that utilize the activity of chromatin remodeling complexes and/or enzymatic chromatin remodeling proteins, would facilitate, among other things, cloning and modulation of cellular differentiation and dedifferentiation.